Benzimidazole-Triazole Hybrids as Antimicrobial and Antiviral Agents: A Systematic Review

Bacterial infections have attracted the attention of researchers in recent decades, especially due to the special problems they have faced, such as their increasing diversity and resistance to antibiotic treatment. The emergence and development of the SARS-CoV-2 infection stimulated even more research to find new structures with antimicrobial and antiviral properties. Among the heterocyclic compounds with remarkable therapeutic properties, benzimidazoles, and triazoles stand out, possessing antimicrobial, antiviral, antitumor, anti-Alzheimer, anti-inflammatory, analgesic, antidiabetic, or anti-ulcer activities. In addition, the literature of the last decade reports benzimidazole-triazole hybrids with improved biological properties compared to the properties of simple mono-heterocyclic compounds. This review aims to provide an update on the synthesis methods of these hybrids, along with their antimicrobial and antiviral activities, as well as the structure–activity relationship reported in the literature. It was found that the presence of certain groups grafted onto the benzimidazole and/or triazole nuclei (-F, -Cl, -Br, -CF3, -NO2, -CN, -CHO, -OH, OCH3, COOCH3), as well as the presence of some heterocycles (pyridine, pyrimidine, thiazole, indole, isoxazole, thiadiazole, coumarin) increases the antimicrobial activity of benzimidazole-triazole hybrids. Also, the presence of the oxygen or sulfur atom in the bridge connecting the benzimidazole and triazole rings generally increases the antimicrobial activity of the hybrids. The literature mentions only benzimidazole-1,2,3-triazole hybrids with antiviral properties. Both for antimicrobial and antiviral hybrids, the presence of an additional triazole ring increases their biological activity, which is in agreement with the three-dimensional binding mode of compounds. This review summarizes the advances of benzimidazole triazole derivatives as potential antimicrobial and antiviral agents covering articles published from 2000 to 2023.

The successive events that occurred from the spring of 2020 until now, regarding the emergence and development of the COVID-19 pandemic, have led the scientific world to investigate more closely the possibility of treating this infectious disease with various antiviral [64][65][66], antimicrobial [67], immunomodulatory [68] or anti-inflammatory [69] drugs, therefore, the discovery of new molecules with simple or hybrid structures, which meet the requirements of the treatment of this condition is absolutely necessary and constitutes the engine for the development of new effective therapeutic agents.
Why did I choose the study of benzimidazole-triazole compounds? Classical drugs containing benzimidazole and triazole rings recommend these heterocycles as essential in building new target compounds with antimicrobial, antiviral, antiparasitic, etc. properties ( Figure 1). In addition, the literature mentions a series of benzimidazole-triazole hybrids with remarkable antimicrobial properties, and antiviral activities, including new anti-SARS-COV-2 agents [70][71][72][73][74], with particular importance in the context of the recent pandemic, which led to the study of synthesis methods, antimicrobial properties, structureproperty relationships, and their biological activities. Therefore, this review aims to provide an update on the synthesis methods of the benzimidazole-triazole hybrids, along with their antimicrobial and antiviral activities, as well as the structure-activity relationship and DFT studies reported in the literature. The advantages of the study of benzimidazole-triazole hybrid compounds refer to a wider range of antimicrobial activities, compared to simple precursor heterocycles, to their better minimum inhibitory concentrations compared to simple component heterocycles, as well as to the need to hire specialized personnel to carry out this research. Therefore, this review aims to provide an update on the synthesis methods of the benzimidazole-triazole hybrids, along with their antimicrobial and antiviral activities, as well as the structure-activity relationship and DFT studies reported in the literature. The advantages of the study of benzimidazole-triazole hybrid compounds refer to a wider range of antimicrobial activities, compared to simple precursor heterocycles, to their better minimum inhibitory concentrations compared to simple component heterocycles, as well as to the need to hire specialized personnel to carry out this research.
The main disadvantages are material because the synthesis of some hybrid compounds requires high costs compared to simple heterocycles, as well as greater time consumption.   with the minimum binding energy (−9.8 and −9.7 kcal/mol) as compared to the standard drug Ciprofloxacin (−7.4 kcal/mol) against the target enzyme DNA gyrase B, as summarized in Figure 2 [122].
ADMET analysis of compounds 8-10 exhibited that they have good absorption properties (%HIA) ranging from 99.57 to 100% [123]. For distribution, the compounds do not permeate the blood-brain barrier (BBB). Moreover, the molecules were negative in the AMES toxicity and carcinogenicity test, which suggests that they are non-mutagenic. Compounds 11a-11g with terminal acetylene and 2-(azidomethoxy)ethyl acetate were condensed using CuI as catalyst and triethylamine (TEA) under microwave irradiation to achieve hybrids 1,2,3-triazole connected via benzene to the benzimidazole nucleus 12a- ADMET analysis of compounds 8-10 exhibited that they have good absorption properties (%HIA) ranging from 99.57 to 100% [123]. For distribution, the compounds do not permeate the blood-brain barrier (BBB). Moreover, the molecules were negative in the AMES toxicity and carcinogenicity test, which suggests that they are non-mutagenic. Compounds 11a-11g with terminal acetylene and 2-(azidomethoxy)ethyl acetate were condensed using CuI as catalyst and triethylamine (TEA) under microwave irradiation to achieve hybrids 1,2,3-triazole connected via benzene to the benzimidazole nucleus 12a-12g with excellent yields (70-90%) (Scheme 2). The cleavage of the acetyl group using potassium carbonate (K 2 CO 3 ) in methanol liberated the hydroxy group of the corresponding hybrid triazoles 13a-13g (Scheme 4). in almost quantitative yields. Compounds 13a-13g were screened for in vitro antifungal activities against two phytopathogenic fungi, Verticillium dahliae Kleb and Fusarium oxysporum f. sp. albedinis. The result of the mycelia linear growth rate indicates that some of the compounds show a weak inhibition against the two fungi, the only compound that shows a significantly increased rate is compound 13e, with a rate of 29.76% against Verticillium dahliae in the sporulation test [124]. Generally, compounds showed better activities against Gram-positive than Gram-negative bacteria. Compounds 20a-20e, with better binding affinity relative to other amidines, were the most active against S. aureus (MIC = 8-32 μgmL −1 ). Compound 19a was the most promising candidate because of its higher potency (MIC = 4 μgmL −1 ) against ESBL-producing E. coli. Results of anti-trypanosomal evaluations showed that p-methoxyphenyl substituent in 19b-21b enhanced activity, with 20b (IC50 = 1.1 mM and IC90 = 3.5 mM) being more potent than Nifurtimox. In contrast to the observed correlation between antimicrobial activity and DNA binding, the antiprotozoal effects of 20b did not correlate with its DNA affinity [125]. Generally, compounds showed better activities against Gram-positive than Gram-negative bacteria. Compounds 20a-20e, with better binding affinity relative to other amidines, were the most active against S. aureus (MIC = 8-32 µgmL −1 ). Compound 19a was the most promising candidate because of its higher potency (MIC = 4 µgmL −1 ) against ESBL-producing E. coli. Results of antitrypanosomal evaluations showed that p-methoxyphenyl substituent in 19b-21b enhanced activity, with 20b (IC 50 = 1.1 mM and IC 90 = 3.5 mM) being more potent than Nifurtimox. In contrast to the observed correlation between antimicrobial activity and DNA binding, the antiprotozoal effects of 20b did not correlate with its DNA affinity [125].
Antibiotics 2023, 12, x FOR PEER REVIEW 7 12g with excellent yields (70-90%)(Scheme 2). The cleavage of the acetyl group using tassium carbonate (K2CO3) in methanol liberated the hydroxy group of the correspon hybrid triazoles 13a-13g (Scheme 4). in almost quantitative yields. Compounds 13a were screened for in vitro antifungal activities against two phytopathogenic fungi, V cillium dahliae Kleb and Fusarium oxysporum f. sp. albedinis. The result of the mycelia li growth rate indicates that some of the compounds show a weak inhibition against the fungi, the only compound that shows a significantly increased rate is compound 13e, a rate of 29.76% against Verticillium dahliae in the sporulation test [124]. Generally, compounds sho better activities against Gram-positive than Gram-negative bacteria. Compounds 20a with better binding affinity relative to other amidines, were the most active against S reus (MIC = 8-32 μgmL −1 ). Compound 19a was the most promising candidate becau its higher potency (MIC = 4 μgmL −1 ) against ESBL-producing E. coli. Results of antianosomal evaluations showed that p-methoxyphenyl substituent in 19b-21b enhance tivity, with 20b (IC50 = 1.1 mM and IC90 = 3.5 mM) being more potent than Nifurtimo contrast to the observed correlation between antimicrobial activity and DNA binding antiprotozoal effects of 20b did not correlate with its DNA affinity [125]. and 26h with MIC of 3.125-6.25 µg mL −1 were found to be the most promising potential antimicrobial molecules [127]. The authors calculated various physicochemical parameters such as clogP, drug score, and drug-likeness of 26a-26j using the Osiris Property Explorer software [128]. For all the compounds, the calculated clogP values were found to be below five according to Lipinski's rule-of-5 and also exhibited positive values for drug score. Mallikanti et al. synthesized novel benzimidazole-conjugated 1,2,3-triazole analogs 29a-29l in two steps: 1. formation of benzimidazole intermediate by reaction between 3',5'-difluorobiphenyl-3,4-diamine 27 and 2-hydroxy-4-(prop-2-ynyloxy) benzaldehyde 28, and 2. microwave-assisted copper-catalyzed click reaction (Scheme 7). Compounds 29a-29l showed minimal inhibition zones against all Gram-positive (S. aureus, B. subtilis) and Gram-negative (E. coli, P. aeruginosa) strains using Ampicillin as A standard drug. Among all tested compounds, the 29i and 29k showed higher activity against P. aeruginosa, S. aureus, and B. subtilis than the standard reference. Compounds 29a, 29b, 29c, 29d, 29e, 29f, 29g, 29h, 29j, and 29l showed moderate antibacterial activity against tested strains (Inhibition zone: 10-25 mm compared with 18-20 mm for Ampicillin). Compounds 29i, 29j, and 29k also established strong activity against both fungal strains, C. albicans MTCC 183 and A. niger MTCC 9652, compared to the standard drug Griseofulvin [70]. To understand the binding mode of novel compounds, docking simulations were performed against the crystal structures of glucosamine-6-phosphate synthase (GlmS) (PDB ID: 2VF5) of E. coli and secreted aspartic proteinase (Sap) 1 (PDB ID: 2QZW) of C. albicans retrieved from the protein data bank. The best active compound, 29l, scored the highest binding affinity value of about −10.0 kal/mol, which demonstrated two key interactions with the active site amino acid Asp549 of Glms with a bond distance of 2.66 and 2.81 Å. Further, the hydrophobic interactions were taken with Tyr312, Ser316, Asp474, Asn523, Ala572, and Ala551 of Glms, among which one π-π T-shaped interaction with Tyr312, and halogen bond [129] interactions with Tyr312, Asn523, and Asn551 ( Figure 4). The binding energies and interactions of all compounds are better than that of Ampicillin, which proves that these molecules could best fit into the cavity of Glms [70]. Chandrika et al. reported hybrids 30-32 with broad spectrum antifungal activity (0.975-3.9 µgmL −1 against C. albicans; 0.12-0.48 µgmL −1 against C. parapsilosis) ( Figure 5). These compounds also displayed good activity against C. albicans biofilms (3.9-15.6 µgmL −1 against C. albicans) [130].

1,2-Bis-Substitutedbenzimidazoles-R(Ar)-1,4-Disubstituted-1,2,3-Triazole.
Rezki reported the intramolecular cyclization of thiosemicarbazides 42a-42d in refluxing aqueous sodium hydroxide (2N) for 6 h with the formation of hybrids 43a-43d with yields of 82-86% (Scheme 11). Among all the 1,2,4-triazole derivatives, N4-phenyl and N4-(4-fluorophenyl) derivatives 43a and 43b were found to be the most potent with MIC values of 4-8 μg mL −1 . Also, triazoles 43c and 43d exerted the best inhibition against both tested fungal strains, A. brasiliensis and Candida albicans, with MIC values ranging from 0.5 to 4 μg mL −1 , more potent than the reference drug Fluconazole. Condensation of compound 44 with several benzaldehydes in refluxing ethanol for 4-6 h with a catalytic amount of HCl produced a new class of hybrid Schiff bases 45a-45g with yields of 84-86% (Scheme 12). The antimicrobial bioassay results for the synthesized Schiff bases 45a-45g revealed that all of the tested compounds were more effective towards all of the organisms, with MIC values of 1-16 μg mL −1 . Among them, Schiff bases 45c, 45d, and 45e with a fluorine atom at position "2" exhibited the highest antibacterial inhibition potency at MIC 1-8 μg mL −1 . The Schiff base 45e containing a CF3 group exerted the highest antifungal inhibition activity with MIC of 1 μg mL −1 [133]. Al-blewi et al. synthesized triazoles 47a-47f in two steps: i. regioselective alkylation of 4 with two equivalents of propargyl bromide in the presence of two equivalents of potassium carbonate as a base catalyst to afford benzimidazole 46 with 91% yield after stirring at room temperature overnight; ii. Copper-mediated Huisgen 1,3-dipolar cycloaddition reaction on compound 46 in good yields (82-88%) (Scheme 13). Generally, bis-1,2,3-triazoles 47a-47f exhibited more potent antimicrobial activities than their mono-1,2,3-triazole derivatives 6a-6f. This was attributed to the synergistic effect of the sulfonamoyl and tethered heterocyclic components in addition to the improved lipophilicity of the bis-substituted derivatives. Among the synthesized compounds, compound 47a was the most potent antimicrobial agent, with MIC values ranging between 32 and 64 μg mL −1 against all tested strains B. cereus, S. aureus, E. coli P. aeruginosa, C. albicans, and A. brasiliensis. Pharmacophore elucidation of the compound 47a-47f was performed based on in silico ADMET evaluation of the tested compounds. Screening results of drug-likeness rules showed that all compounds follow the accepted rules, meet the criteria of drug-likeness, and follow Lipinski's rule of five. In addition, the toxicity results showed that all compounds are non-mutagenic and noncarcinogenic [119]. revealed that all of the tested compounds were more effective towards all of the organisms, with MIC values of 1-16 µg mL −1 . Among them, Schiff bases 45c, 45d, and 45e with a fluorine atom at position "2" exhibited the highest antibacterial inhibition potency at MIC 1-8 µg mL −1 . The Schiff base 45e containing a CF 3 group exerted the highest antifungal inhibition activity with MIC of 1 µg mL −1 [133]. Al-blewi et al. synthesized triazoles 47a-47f in two steps: i. regioselective alkylation of 4 with two equivalents of propargyl bromide in the presence of two equivalents of potassium carbonate as a base catalyst to afford benzimidazole 46 with 91% yield after stirring at room temperature overnight; ii. Copper-mediated Huisgen 1,3-dipolar cycloaddition reaction on compound 46 in good yields (82-88%) (Scheme 13). Generally, bis-1,2,3-triazoles 47a-47f exhibited more potent antimicrobial activities than their mono-1,2,3-triazole derivatives 6a-6f. This was attributed to the synergistic effect of the sulfonamoyl and tethered heterocyclic components in addition to the improved lipophilicity of the bis-substituted derivatives. Among the synthesized compounds, compound 47a was the most potent antimicrobial agent, with MIC values ranging between 32 and 64 µg mL −1 against all tested strains B. cereus, S. aureus, E. coli P. aeruginosa, C. albicans, and A. brasiliensis. Pharmacophore elucidation of the compound 47a-47f was performed based on in silico ADMET evaluation of the tested compounds. Screening results of drug-likeness rules showed that all compounds follow the accepted rules, meet the criteria of drug-likeness, and follow Lipinski's rule of five. In addition, the toxicity results showed that all compounds are non-mutagenic and noncarcinogenic [119].    Aparna et al. used a similar strategy for obtaining nine new bis-1,2,3-triazol-1Hsubstituted arylbenzimidazole-2-thiol derivatives 48a-48l ( Figure 6). Antibacterial act of triazole derivatives 48 demonstrates moderate to good activity against Gram-neg (E. coli, S. typhy, P. aeruginosa) and Gram-positive (S. aureus) bacterial strains. The prod 48i, 48k, and 48l are characterized by a broad spectrum of antibacterial activity at a centration of 10 μg mL −1 . The synthesized 1,2,3 triazole derivatives were studied for molecular docking on the high-resolution X-ray crystal structure of FabI of Staphyloc aureus (pdb id:4FS3) obtained from the protein data bank [134]. The highest dock sco -7.69 kcal/mol and the lowest dock score of -0.942 kcal/mol were obtained for molec 48l and 48h, respectively [135].  Aparna et al. used a similar strategy for obtaining nine new bis-1,2,3-triazol-1H-4-ylsubstituted arylbenzimidazole-2-thiol derivatives 48a-48l ( Figure 6). Antibacterial activity of triazole derivatives 48 demonstrates moderate to good activity against Gram-negative (E. coli, S. typhy, P. aeruginosa) and Gram-positive (S. aureus) bacterial strains. The products 48i, 48k, and 48l are characterized by a broad spectrum of antibacterial activity at a concentration of 10 μg mL −1 . The synthesized 1,2,3 triazole derivatives were studied for their molecular docking on the high-resolution X-ray crystal structure of FabI of Staphylococcus aureus (pdb id:4FS3) obtained from the protein data bank [134]. The highest dock score of -7.69 kcal/mol and the lowest dock score of -0.942 kcal/mol were obtained for molecules 48l and 48h, respectively [135].

Benzimidazole-R(Ar)-1,2,3-Triazole Hybrids as Antitubecular Agents
Ashok reported compound 26h has the best antitubercular drug candidate by inhibiting the growth of the MTB (Mycobacterium tuberculosis) strain with MIC = 3.125 µ mL −1 (7.1 µM) (control Rifampicin MIC = 0.04 µg mL −1 and isoniazid MIC = 0.38 µg mL −1 ). The best antitubercular activity of 26h may be attributed to the presence of the nitro group on the phenyl ring at the ortho position. Compound 26b (MIC = 6.25 µg mL −1 (14.7 µM)) with chlorine substituent, compound 26i (MIC = 6.25 µg mL −1 (14.2 µM)) with trifluoromethyl substituent and compound 26j (MIC = 12.5 µg mL −1 (28.4 µM)) with benzyl substituent exhibited moderate antitubercular activity. Therefore, the incorporation of the electronwithdrawing nitro group, electronegative chlorine, and trifluoromethyl groups on the phenyl ring was highly favored for antitubercular activity. The authors calculated various physicochemical parameters and found from the theoretical data that compounds 26a-26j also exhibited positive values for drug score [127]. Gill et al. reported syntheses of hybrids 51a-51d by reaction between 2-(3-fluorophenyl)-1H-benzo[d]imidazole 50 and phenylsubstituted 4-(bromomethyl)-1-phenyl-1H-1,2,3-triazole 49 in DMF at room temperature (Scheme 14). Trifluorosubstituted-compound 51a possessed enhanced anti-mycobacterial activity, >96% of inhibition at 6.25 µg concentration. Also, compounds 51b and 51c, which had antimicrobial activities superior to the other compounds, were reported as the best choice for the preparation of new derivatives in order to improve effectiveness on intracellular mycobacteria (macrophage) or in infected animals [136]. Anand et al. reported a one-pot reaction between 2-propargylthiobenzimidazole 4, 4-bromomethylcoumarins/1aza-coumarins 52/53 and sodium azide under click chemistry conditions to give exclusively 1,4-disubstituted triazoles 54a-54n. (Scheme 15). Antitubercular assays against M. tuberculosis (H37Rv) coupled with in silico molecular docking studies indicated that dimethyl substituents 54c and 54d showed promising activity (MIC = 3.8 µMol L −1 ) with higher C-score values. Surflex-Dock was used to investigate detailed intermolecular interactions between the ligand and the target protein. Three-dimensional structure information on the target protein was taken from the PDB entry 4FDO. Processing of the protein included the removal of the co-crystallized ligand and water molecules, as well as the addition of essential hydrogen atoms. All 14 inhibitors 54a-54n were docked into the active site of ENR, as shown in Figure 7a, and Figure 7b indicates the superimposition of compounds 54a and 54d with ligand [137]. Khanapurmath et al. synthesized triazoles 55 by click reaction (Figure 8a). Benzimidazolone bis-triazoles 55a-55n showed better activity with MIC in the range 2.33-18.34 µM, and the most active compounds were 55h and 55m. All compounds exhibited moderate to low levels of cytotoxicity with IC 50 values of the human embryonic kidney cells in the range of 943-12294 µM, and none of the 14 compounds exhibited any significant cytotoxic effects, suggesting huge potential for their in vivo use as antitubercular agents. Docking studies revealed an additional interaction of benzimidazolone oxygen in these compounds (Figure 8b) [138]. Also, Sharma et al. summarize 1,2,3-triazoles as antitubercular compounds and various hybrids with benzimidazole, coumarin, isoniazid, quinolines, etc. [39].

2-Benzimidazole-R(Ar)-1-(1,2,4-Triazole)
Pandey et al. synthesized hybrids 59a-59e in three steps: reaction of 7-hydroxy-4methyl coumarin with thiosemicarbazide to form triazole intermediate 57, which underwent Mannich reaction with formaldehyde, and an amino acid to form intermediates 58a-  . The antifungal activity of compound 73 was tested against Candida albicans spores in vitro (turbidimetric method) and in vivo (kidney burden test). Compound 73i had a good antifungal activity as compared with the other twelve compounds at 0.0075 μM mL −1 , which is equivalent to Fluconazole activity both in vitro and in vivo. The antifungal activity decreased with the increasing alkyl length of N1 of benzimidazole (methyl to ethyl). This was proven for compounds 73i and 73l, which showed MICs of 0.0075 and 0.015 μM mL −1 , respectively. The ligand fit method was performed to study and predict the binding mode of the hybrids 73 with the target enzyme (homology modeled) cytochrome P450 lanosterol 14-α-demethylase of C. albicans. All compounds showed binding in the active site of the enzyme. The 1,2,4-triazole ring of compounds 73a-73l is positioned almost perpendicular to the porphyrin plane, with a ring nitrogen (N-4) atom coordinated to the heme iron ( Figure 10) [145,146]. Ahuja et al. reported antifungal activity of compounds 74a-74c against F. verticillioides, D. oryzae, C. lunata, and F. fujikuroi ( Figure 11). All compounds had increased potency than the standard commercial benzimidazole fungicide, carbendazim (Table 5).  Pandey et al. synthesized hybrids 59a-59e in three steps: reaction of 7-hydroxy-4methyl coumarin with thiosemicarbazide to form triazole intermediate 57, which underwent Mannich reaction with formaldehyde, and an amino acid to form intermediates 58a-58e. Intermediates 58a-58e reacted with o-phenylenediamine in pyridine to give benzimidazole-1,2,4-triazole hybrids in poor yields (Scheme 16). Compound 59a displayed promising antifungal activity against Candida albicans and Cryptococcus himalayensis since the MIC value in each case was found to be 3.5 µg mL -1 . Compound 59b showed low to moderate antifungal activity against all five fungi, Candida albicans, Cryptococcus himalayensis, Sporotrichum schenkii, Trichophyton rubrum, and Aspergillus fumigatus [139].
58e. Intermediates 58a-58e reacted with o-phenylenediamine in pyridine to give benzimidazole-1,2,4-triazole hybrids in poor yields (Scheme 16). Compound 59a displayed promising antifungal activity against Candida albicans and Cryptococcus himalayensis since the MIC value in each case was found to be 3.5 μg mL -1 . Compound 59b showed low to moderate antifungal activity against all five fungi, Candida albicans, Cryptococcus himalayensis, Sporotrichum schenkii, Trichophyton rubrum, and Aspergillus fumigatus [139].   17). All compounds were screened for antimicrobial activity against different Gram-positive organisms, S. aureus, P. aeruginosa, and Gram-negative organisms, E. coli and S. typhosa using Gentamycin as a reference standard. The data generated from preliminary screening showed that compounds displayed moderate to better antimicrobial activity. Compounds 63a, 63e, 63f, 63h, 63i, and 63l displayed maximum activity (Table 4) [140].  All compounds were screened for antimicrobial activity against different Gram-positive organisms, S. aureus, P. aeruginosa, and Gram-negative organisms, E. coli and S. typhosa using Gentamycin as a reference standard. The data generated from preliminary screening showed that compounds displayed moderate to better antimicrobial activity. Compounds 63a, 63e, 63f, 63h, 63i, and 63l displayed maximum activity (Table 4) [140].  (Figure 9). From the antifungal activity data, preliminary SARs was obtained. In general, the amine linker was important for antifungal activities. Substituted piperazine derivatives were comparable or superior to the corresponding N-methyl derivatives. [142]. Luo et al. reported a series of naphthalimide benzimidazole-1,2,4-triazole hybrids 68a-68h and the corresponding triazolium salts 69a-69d prepared by convenient and efficient procedures starting from naphthalimide triazole 66 (Scheme 18). 2-Chlorobenzyl triazolium 68g and compound 69b with octyl group exhibited the best antibacterial activities among all the tested compounds, especially against S. aureus with an inhibitory concentration of 2 μg mL −1 which was equipotent potency to Norfloxacin (MIC = 2 μg mL −1 ) and more active than Chloromycin (MIC= 7 μg mL −1 ). Triazoliums 68g and 68f bearing 3-fluorobenzyl moiety displayed the best antifungal activities (MIC = 2-19 μg mL −1 ) against all the tested fungal strains, C. albicans ATCC 76615, A. fumigatus ATCC 96918, C. utilis, S. cerevisia and A. flavus, without being toxic to PC12 cell line within concentration of 128 μg mL −1 . Further investigations showed that compound 68g could intercalate into calf thymus DNA to form the 68g-DNA complex, which could block DNA replication, exerting powerful antimicrobial activities. [143]. Benzimidazole-1,2,4-triazole Mannich base 70 was active against Bacillus subtilis and Bacillus pumilus (inhibition zone diameters being 19 and 17 mm, respectively, compared Scheme 17. Synthesis of benzimidazole-1,2,4-triazoles 63a-63e.  (Figure 9). From the antifungal activity data, preliminary SARs was obtained. In general, the amine linker was important for antifungal activities. Substituted piperazine derivatives were comparable or superior to the corresponding N-methyl derivatives. [142]. Luo et al. reported a series of naphthalimide benzimidazole-1,2,4-triazole hybrids 68a-68h and the corresponding triazolium salts 69a-69d prepared by convenient and efficient procedures starting from naphthalimide triazole 66 (Scheme 18). 2-Chlorobenzyl triazolium 68g and compound 69b with octyl group exhibited the best antibacterial activities among all the tested compounds, especially against S. aureus with an inhibitory concentration of 2 µg mL −1 which was equipotent potency to Norfloxacin (MIC = 2 µg mL −1 ) and more active than Chloromycin (MIC = 7 µg mL −1 ). Triazoliums 68g and 68f bearing 3-fluorobenzyl moiety displayed the best antifungal activities (MIC = 2-19 µg mL −1 ) against all the tested fungal strains, C. albicans ATCC 76615, A. fumigatus ATCC 96918, C. utilis, S. cerevisia and A. flavus, without being toxic to PC12 cell line within concentration of 128 µg mL −1 . Further investigations showed that compound 68g could intercalate into calf thymus DNA to form the 68g-DNA complex, which could block DNA replication, exerting powerful antimicrobial activities. [143]. Benzimidazole-1,2,4-triazole Mannich base 70 was active against Bacillus subtilis and Bacillus pumilus (inhibition zone diameters being 19 and 17 mm, respectively, compared to Ciprofloxacin with 28 and 30 mm, respectively) [144].   . The antifungal activity of compound 73 was tested against Candida albicans spores in vitro (turbidimetric method) and in vivo (kidney burden test). Compound 73i had a good antifungal activity as compared with the other twelve compounds at 0.0075 µM mL −1 , which is equivalent to Fluconazole activity both in vitro and in vivo. The antifungal activity decreased with the increasing alkyl length of N1 of benzimidazole (methyl to ethyl). This was proven for compounds 73i and 73l, which showed MICs of 0.0075 and 0.015 µM mL −1 , respectively. The ligand fit method was performed to study and predict the binding mode of the hybrids 73 with the target enzyme (homology modeled) cytochrome P450 lanosterol 14-α-demethylase of C. albicans. All compounds showed binding in the active site of the enzyme. The 1,2,4triazole ring of compounds 73a-73l is positioned almost perpendicular to the porphyrin plane, with a ring nitrogen (N-4) atom coordinated to the heme iron ( Figure 10) [145,146]. Ahuja et al. reported antifungal activity of compounds 74a-74c against F. verticillioides, D. oryzae, C. lunata, and F. fujikuroi ( Figure 11). All compounds had increased potency than the standard commercial benzimidazole fungicide, carbendazim (Table 5).   Compound 74c exhibited ED50 values lower than triazole fungicide, propiconazole The results reinforced the synergistic effects of the benzimidazole and 1,2,4-triazole com bination supported by a computational approach. Hydrogen bonding interactions were more pronounced in compounds 74a-74c in the binding pockets of both the target enzymes, in comparison to standards. In compound 74c, two H-bonds were formed with   Compound 74c exhibited ED50 values lower than triazole fungicide, propiconazole. The results reinforced the synergistic effects of the benzimidazole and 1,2,4-triazole combination supported by a computational approach. Hydrogen bonding interactions were more pronounced in compounds 74a-74c in the binding pockets of both the target enzymes, in comparison to standards. In compound 74c, two H-bonds were formed with Gln11 present in the binding cleft of the active pocket of β-tubulin ( Figure 12). In all three compounds, the right position of the N-atoms of both 1,2,4-triazole and benzimidazole, that O-atoms of methoxy and carbonyl groups, contributed well to strong binding into the   Compound 74c exhibited ED50 values lower than triazole fungicide, propiconazole. The results reinforced the synergistic effects of the benzimidazole and 1,2,4-triazole combination supported by a computational approach. Hydrogen bonding interactions were more pronounced in compounds 74a-74c in the binding pockets of both the target enzymes, in comparison to standards. In compound 74c, two H-bonds were formed with Gln11 present in the binding cleft of the active pocket of β-tubulin ( Figure 12). In all three  Compound 74c exhibited ED 50 values lower than triazole fungicide, propiconazole. The results reinforced the synergistic effects of the benzimidazole and 1,2,4-triazole combination supported by a computational approach. Hydrogen bonding interactions were more pronounced in compounds 74a-74c in the binding pockets of both the target enzymes, in comparison to standards. In compound 74c, two H-bonds were formed with Gln11 present in the binding cleft of the active pocket of β-tubulin ( Figure 12). In all three compounds, the right position of the N-atoms of both 1,2,4-triazole and benzimidazole, that O-atoms of methoxy and carbonyl groups, contributed well to strong binding into the active site of enzymes via H-bonding [147]. Evren  . Although the antibacterial activities of compounds 79a-79c against Escherichia coli ATCC 35218, E. coli ATCC 25922, Klebsiella pneumoniae NCTC 9633, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 13311, and Staphylococcus aureus ATCC 25923, were weak, the antifungal activities against C. albicans were found promising, with MIC values of 3.9, 7.8, and 3.9 µg mL −1 respectively, using as reference drug Ketokonazole (MIC = 7.8 µg mL −1 ). Protein-ligand interactions and binding poses of the 79a, 79b, and 79c compounds on the CYP51 active site were examined. As shown in Figure 13, an H bond of 2.11 Å between compound 79a and Met508, π-π stacking interactions with Tyr118, Hie377, Phe233, generated hydrophobic interactions with Pro230, Leu376, Tyr64, Phe228, and Tyr505. Theoretical ADME calculations of the 79a, 79b, and 79c were made, and the compounds were found to have good lipophilicity, moderate water solubility, and within the limiting rules of Lipinski, Ghose, Veber, Egan, and Muegge ( Figure 14) [148]. Ghobadi et al. reported the synthesis of compounds 85a-85e, in two different ways, from 3,4-diaminobenzophenone 80, i. formation of 2-mercapto benzimidazole derivatives 82, 83, and ii. nucleophilic ring opening of various oxiranes 84a-84e with benzimidazoles 82 and 83 using NaHCO 3 in ethanol at room temperature (Scheme 21). Compounds 85a-85e, containing a 5-benzoylbenzimidazole scaffold, showed better antifungal activity against Candida spp. and Cryptococcus neoformans than related benzimidazole and benzothiazole derivatives. The better results were obtained with 4-chloro-derivative 85b displaying MICs < 0.063-1 µg mL −1 . Also, compound 86c, synthesized analogously, is as potent as compound 85b. The docking experiments were conducted to further rationalize the obtained antifungal activity data and investigate the type of interactions between compound 85b and the active site of lanosterol 14α-demethylase (CYP51). As shown in Figure 13, the coordinated bond-forming distance between the N4 atom of the triazole nucleus of compound 85b and the iron atom in the heme group of active site were 2.71 and 2.40 Å, respectively. A hydrogen-bonding interaction between Tyr132 and the sulfur group of (S)-85b was observed. In vitro and in silico ADMET evaluations of the most promising compounds 85b indicated that the selected compounds have desirable ADMET properties in comparison to the standard drug Fluconazole. A docking simulation study demonstrated that the benzimidazol-2-yl-thio moiety is responsible for the potent antifungal activity of these compounds [72].        Table 8 [153]. El-masry et al. synthesized compounds 98 and 99 and found that they did not exhibit antimicrobial activity ( Figure 16) [154]. Menteşe et al. synthesized compounds 100a-100d, for which they found no antimicrobial activity on the ten strains tested [155]. Karale et al. synthesized bis-benzimidazole-1,2,4-triazole hybrids 102a-102e (Scheme 25) in four steps from 7-methyl-2-propyl-3H-benzo[d]imidazole-5-carboxylic acid. All compounds 102 did not show antimicrobial activity against the strains tested, C. albicans, A. fumigatus, S. aureus, and E. coli [156,157].

2-Benzimidazole-R(Ar)-2-1,2,4-Triazole
Eisa et al. synthesized compounds 105a and 105b (Scheme 26) (Table 9) Table 10 reveal that compounds 109a, 109c, and 109e exhibited satisfactory effects against S.aureus and E.coli, while compounds 109b, 109f, and 109g showed moderate activity against the same microbes. Also, the antifungal activity of these compounds was screened against Candida albicans. Compounds 109a and 109d showed the highest degree of inhibition against C.albicans when compared with the standard drug Ketoconazole [159]. Can [160]. Gencer et al. synthesized compounds 112 in good yields (77-88%) using a similar strategy (Figure 17). Microbiological studies revealed that compounds 112a, 112b, 112c, 112e, 112f, 112g, and 112h possess a good antifungal profile against all tested strains, C. albicans, C. glabrata, C. krusei, C. parapsilopsis, with MIC50 = 0.78-1.56 μg mL −1 . Compound 112i was the most active derivative and showed comparable antifungal activity to those of reference drugs Ketoconazole and Fluconazole [161]. The SAR (Structure-activity relationship) on the synthesized benzimidazole-triazole compounds is summarized in Figure 18. It is observed that the presence of chlorine or fluorine in the "5" position of benzimidazole, as well as the presence of fluorine in the "4" position of phenyl, increase the antibacterial activity, while the presence of fluorine in the "2" position of phenyl does not change the activity, and the    (Table 9) by the reaction between 2-(chloromethyl)-1H-benzo[d]imidazole 103 and 4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazole-3-thiol 104a or 4-phenyl -5-(thiophen-2-yl)-4H-1,2,4-triazole-3-thiol 104b, at reflux in absolute ethanol, for 12 h. Also, they reported synthesis of the compounds 107a and 107b from 2-(2-(phenylthiomethyl)-1H-benzo[d]imidazol-1-yl)acetohydrazide in two steps (Scheme 27). All compounds showed antimicrobial activity against Escherichia coli superior to that of standard Gentamicin. Compound 107a exhibited only moderate activity against Staphylococcus aureus [158]. Nevade et al. synthesized compounds 109a-109h in five steps from 1H-benzo[d]imidazole-2-thiol 108 (Scheme 28). The antimicrobial screening results presented in Table 10 reveal that compounds 109a, 109c, and 109e exhibited satisfactory effects against S.aureus and E.coli, while compounds 109b, 109f, and 109g showed moderate activity against the same microbes. Also, the antifungal activity of these compounds was screened against Candida albicans. Compounds 109a and 109d showed the highest degree of inhibition against C.albicans when compared with the standard drug Ketoconazole [159]. Can et al. synthesized hybrids 111a-111h in four steps from methyl 4-(5-methyl-1H-benzo[d]imidazol-2-yl)benzoate 110 (Scheme 29). All compounds were screened for antifungal activity against Candida albicans ATCC 24433, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22,019 (Table 11). Compounds 111i and 111s exhibited significant inhibitory activity against Candida strains with MIC 50 values ranging from 0.78 to 1.56 µg mL −1 [160]. Gencer et al. synthesized compounds 112 in good yields (77-88%) using a similar strategy (Figure 17). Microbiological studies revealed that compounds 112a, 112b, 112c, 112e, 112f, 112g, and 112h possess a good antifungal profile against all tested strains, C. albicans, C. glabrata, C. krusei, C. parapsilopsis, with MIC 50 = 0.78-1.56 µg mL −1 . Compound 112i was the most active derivative and showed comparable antifungal activity to those of reference drugs Ketoconazole and Fluconazole [161]. The SAR (Structure-activity relationship) on the synthesized benzimidazole-triazole compounds is summarized in Figure 18. It is observed that the presence of chlorine or fluorine in the "5" position of benzimidazole, as well as the presence of fluorine in the "4" position of phenyl, increase the antibacterial activity, while the presence of fluorine in the "2" position of phenyl does not change the activity, and the presence of groups CH 3 or C 2 H 5 in position "4" in the triazole nucleus does not bring any change in the antibacterial activity of the compounds. Furthermore, toxicological and ADME studies indicated the relative potency of hybrids 112h and 112i, according to the literature [162][163][164][165][166]. Compound 112i also inhibited ergosterol biosynthesis concentration dependently. Results of ergosterol level quantification assay and fluorescence microscopy studies revealed that the mechanism of action of hybrids is associated with the inhibition of ergosterol biosynthesis, which may subsequently result in altered membrane fluidity, plasma membrane biogenesis, and functions of fungi. Güzel et al. synthesized a new series of benzimidazole-1,2,4-triazole derivatives 113a-113l using the same procedure described in Scheme 29 as potential antifungal agents ( Figure 19). All the compounds were screened for their in vitro antifungal activity against four fungal strains, namely, C. albicans, C. glabrata, C. krusei, and C. parapsilopsis and were found to exhibit excellent activity against C. glabrata. Especially, compounds 113b, 113i, and 113j were found to be the most effective compounds in the series with an MIC value of 0.97 µg mL −1 [71]. According to the molecular docking study, compounds 113b, 113i, and 113j fit into the LDM enzyme active pocket. In a previous study [167], the Tyr118 amino acid and HEM601 protein were described as essential residues, and in this study, the synthesized active compounds interacted significantly with Tyr118, His377, and HEM601 residues. The interactions with HEM were seen as π−π stacking and π−cation interactions. Therefore, the antifungal effects of compounds 113b, 113i, and 113j were considered to be caused by the destruction of cell integrity due to the inhibition of the LDM enzyme. The authors identified compound 6i with higher inhibitory activity due to H-bonding with Tyr132, unlike the other two compounds. Aryal et al. reported synthesis of 2-substituted benzimidazole containing 1,2,4-triazoles 114a and 114b ( Figure 20). The compounds did not show antimicrobial activity against the tested strains Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis ATCC 1228 [168]. Kazeminejad et al. did a study on 1,2,4-triazoles as well as structure-activity relationships (SAR) [38].      Cl and F enhance activity F did not change the activity F significantly increaseas the activity Not differences between CH 3 and C 2 H 5 Figure 18. SAR outline of the benzimidazole-1,2,4-triazole hybrids 112a-112i.     Figure 19. Structure of benzimidazole-1,2,4-triazole hybrids 113a-113l.

Synthesis and Antiviral Activities of Benzimidazole-Triazoles
Over 200 viruses are known to cause disease in humans, yet currently approved antiviral drugs are available to treat only about 10 of these viral infections [170,171]. The past decade has underscored the global threat posed by emerging viruses. An alternative solution is the development of broad-spectrum antiviral drugs. One advantage of this approach is reduced time and cost associated with the early stages of drug development per approved indication. It can also diminish the clinical risks in more advanced stages of development [172,173]. Youssif

Synthesis and Antiviral Activities of Benzimidazole-Triazoles
Over 200 viruses are known to cause disease in humans, yet currently approved antiviral drugs are available to treat only about 10 of these viral infections [170,171]. The past decade has underscored the global threat posed by emerging viruses. An alternative solution is the development of broad-spectrum antiviral drugs. One advantage of this approach is reduced time and cost associated with the early stages of drug development per approved indication. It can also diminish the clinical risks in more advanced stages of development [172,173]. Youssif et al. reported the synthesis of benzimidazole-1,2,3-triazole hybrids 2-{4-[(1-benzoylbenzimidazol-2-ylthio)methyl]-1H-1,2,3-triazol-1-yl}-N-(4nitro--phenyl)-acetamide 116 and 2-(4-{[1-(4-chlorobenzoyl)-benzimidazol-2-ylthio)methyl]-1H-1,2,3-triazol -1-yl}-N-(4-nitrophenyl)-acetamide 117 which showed significant activity against hepatitis C virus (HCV) ( Figure 21). Thus, fifty percent effective concentrations (EC50) of HCV inhibition for compounds 116 and 117 were 7.8 and 7.6 μmol L -1 , respectively, and the 50% cytotoxic concentrations (CC50) were 16.9 and 21.1 μmol L -1 . The results gave an insight into the importance of the substituent at position 2 of benzimidazole for the inhibition of HCV [73]. The antiviral activity of compounds 59a-59e was tested against two viruses, viz., Japanese encephalitis virus (JEV) (P20778), an RNA virus of higher pathogenicity, and Herpes The antiviral activity of compounds 59a-59e was tested against two viruses, viz., Japanese encephalitis virus (JEV) (P20778), an RNA virus of higher pathogenicity, and Herpes simplex virus type-I (HSV-I) (753166), the most common virus present in the environment. The antiviral activity of the compounds data is given in Table 12. All but one of the five compounds were found active against JEV. Compound 59b displayed 90% CPE (cytopathic effect) in vitro with an effective concentration of 8 µg mL -1 , while in vivo activity was less significant (16% protection with an MST of 4 days). The authors suggested that these compounds are better anti-JEV agents than anti-HSV agents since two such compounds, namely 59b and 59e, also displayed a measurable degree of anti-JEV activity in vivo. Compound 59c was found antivirally inactive against both viruses. The anti-HSV-I activity was found to be in the order of 33, 46, 53, and 64% for compounds 59a, 59b, 59d, and 59e, respectively. Since among compounds 59a to 59e, only compound 59e contains a methyl group instead of H as R 1 ; it follows that R 1 does not seem to be responsible for the biological activity [139].   (Table 13). Though not particularly impressive, the presently uncovered activity against BVDV, YFV, and CVB2 is of some interest because it may lead, through the identification of the target, to the development of broad spectrum antiviral agents. In this respect, the definition of the mode of action of the above compounds is mandatory. Furthermore, since the activity against these viruses was influenced by the presence and nature of the substituents in position "5" of the benzimidazole ring, it will be worthwhile to further explore the effect of diversified substitutions as a possibility to improve activity and/or decrease cytotoxicity [174]. SARS-CoV-2 and its variants, especially the Omicron variant, remain a great threat to human health [10]. More novel variants of SARSCoV-2 are also expected to originate in the future. Therefore, efforts should be made to develop wideranging measures to prevent future outbursts of zoonotic origin. Recent articles reported essential and up-to-date information about SARS-CoV-2 variants, antiviral drugs, and vaccines used to fight it [175,176]. information about SARS-CoV-2 variants, antiviral drugs, and vaccines used to fight it [175,176].  Figure 22. Structure of antiviral benzimidazole-1,2,3-triazole hybrids 118-137. Table 13. RSV, BVDV, YFV, and CVB2 Inhibitory Activity of hybrids 118-137 expressed as EC50 (μM).

Compound Anti-RSV Activity Anti-BVDV Activity
Anti-YFV Activity Al-Humaidi et al. reported the synthesis of a series of benzimidazole-1,2,3-triazoles 138-140 ( Figure 23). Molecular docking studies and in vitro enzyme activity revealed that most of the investigated compounds demonstrated promising binding scores against the SARS-CoV-2 and Omicron spike proteins in comparison to the reference drugs (Table 14).  Table 13. RSV, BVDV, YFV, and CVB2 Inhibitory Activity of hybrids 118-137 expressed as EC 50 (µM).

Compound
Anti-RSV Activity

Figure 24.
Three-dimensional binding mode of compound 140 (green) at the binding interface between the Omicron S-RBD (red) and human ACE2 (blue) [74]. Figure 24. Three-dimensional binding mode of compound 140 (green) at the binding interface between the Omicron S-RBD (red) and human ACE2 (blue) [74].

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The presence of the orthoor para-substituted phenyl substituent in the "1" position of 1,2,3-triazoles in benzimidazole-triazole hybrids can increase their antimicrobial activity. - In the case of benzimidazoles substituted in the "1" position with triazoles, the presence of an aliphatic or aromatic radical substituent increases the antimicrobial activity of the hybrids. - The presence of the oxygen atom in the bridge that connects the benzimidazole and triazole rings is favorable to the antimicrobial activity of the hybrids (compounds 19, 20, 21, 29, 30).

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The presence of the sulfur atom in the bridge that connects the benzimidazole and triazole rings is favorable to the antimicrobial activity of the hybrids and even to the antitubercular activity (95-97, 105, 107).

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The phenyl nucleus as a spacer between the "1" position of 1,2,4-triazole and the "2" position of benzimidazole favors the formation of antimicrobial compounds, and the substituents in the "5" position of the benzimidazole nucleus increase the antimicrobial activity (compounds 79, 111, 112, 113). The presence of both the benzimidazole ring and the triazole ring in a single molecule enhanced the effectiveness of the antimicrobial activities, as seen in the sections above. The recent ADME and SAR studies mentioned in this review are also important for directing new syntheses of benzimidazole-triazole hybrids in close correlation with their properties.
As mentioned in the cited literature, it is extremely useful, both from a therapeutic and economic point of view, that the synthesized compounds, such as the benzimidazoletriazole hybrids analyzed in this review, possess both antimicrobial and antimicrobial biological activity antiviral, to meet the medical requirements demanded especially lately, for better action, especially in the case of SARS-CoV-2.
The ADME studies performed on the benzimidazole-triazole hybrids mentioned in this review recommend the compounds as antimicrobials and antivirals and open new horizons to create new compounds, following the conclusions found here, with improved biological properties.
The articles researched on this topic, although they report the general characteristics of these molecules (lipophilicity/hydrophilicity), in order to have the desired antimicrobial or antiviral properties, refer only to liquid formulations in the form in which the compounds were tested, and so far not no article is reported that formulates in the form of nanosystems, nanoparticles for better availability of the active substance. This remains an open research topic for future studies.
We hope that this review will be useful for the design and synthesis of new benzimidazoletriazole hybrids with antimicrobial and antiviral properties in the context of exacerbation of microbial and viral infections and resistance to treatments with drugs known on the market.